P1003: PERIPHERAL BLOOD CYTOTOXIC T CELLS SHOW EARLY EXHAUSTED FEATURES IN MYELOFIBROSIS PATIENTS
Authors: L. Tavernari, S. Rontauroli, M. Maccaferri, B. Mora, E. Bianchi, S. Parenti, E. Genovese, P. Guglielmelli, C. Carretta, S. Mallia, M. Mirabile, S. Sartini, C. Colasante, L. Potenza, F. Passamonti, E. Tagliafico, M. Luppi, A. M. Vannucchi, R. Manfredini
Journal: HemaSphere (2022)

Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by megakaryocyte hyperplasia, bone marrow fibrosis and extramedullary hematopoiesis. Compelling evidence are suggesting that persistent antigen stimulation in the tumor microenvironment can differentiate T effector cells into terminally exhausted T cells, a functional state characterized by decrease in proliferation, cytotoxicity, and cytokine production. Cutting-edge therapies are focused on the reversion of exhausted state through immune checkpoint inhibition to recover the immune response against the tumor. Aims: Despite MPNs are myeloid neoplasms, T cells are showing dysfunctional features. To assess if immune checkpoint inhibition could be a new effective therapy in combination with those currently approved, we investigated T cell exhaustion in MF. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 38 MF patients and 20 healthy donors (HD). The expression profile of the following inhibitory receptors (IRs): PD1, CD244, CD160, TIM3, LAG3, CTLA4 was characterized by flow cytometry in CD3+CD8+ T cells. Results were correlated with clinical features such as Dynamic International Prognostic Scoring System (DIPSS) classification, splenomegaly, hemoglobin level and other negative prognostic factors. For T cell activation assays we stimulated PBMCs overnight in the presence of coated anti-CD3, anti-CD28 and Brefeldin A, whilst cytokine production was then assessed by intracellular flow cytometry staining. Chi-squared and Mann Whitney tests were used for statistical analysis. Total cellular RNA was isolated from peripheral blood granulocytes of 141 primary and secondary MF patients and 28 HDs, and we performed Gene expression profiling (GEPs) by microarray platform. Results: We reported expansion of PD1+CD244+, PD1+CD160+, PD1+CD57- cytotoxic T cell populations from MF patients, together with increased expression of all the IRs assessed on both total CD3+CD8+ T cells and PD1+CD57- T cells fraction. Furthermore, we observed decreased secretion of IFNɣ and TNFɑ by CD3+CD8+ T cells of MF patients, while significant reduction of Granzyme B production was observed mainly in patients with expansion of PD1+CD57- T cell population. GEPs analysis highlighted increased expression of IR-ligands like CD274, CEACAM1, CD48 on granulocytes from MF patients compared to HDs. The correlation analysis of IRs expression with patients’ clinical features showed that higher levels of PD1, CD244, CD160, CTLA4, LAG3 or TIM3 associates with detrimental features like splenomegaly, low hemoglobin levels, HMR mutation number and more severe DIPSS classification. Summary/Conclusion: Our data evidenced in MF patients the presence of an impaired population of peripheral blood cytotoxic T cells expressing multiple IRs and with reduced cytokine production after in vitro activation. Moreover, granulocytes from MF patients display higher level of IR-ligands transcripts hinting an immunosuppressive interplay between the myeloid neoplastic clone and T cells. Lastly, clinical correlations suggest a more severe disease in patients with higher IRs expression on cytotoxic T cells. Taken together this data highlights an early exhausted state likely implicated in immune escape which will be investigated for immune checkpoint inhibition.